Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in Zoysia japonica

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N-6 medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01-0.02 mg L-1 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L-1 2,4-D, 0.5 mg L-1 kinetin, 500 mg L-1 casein hydrolysate, 500 mg L-1 proline, and 500 mg L-1 myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of > 90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO4 to the subculture media maintained > 90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.